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ARTICLE IN PRESSstress response and cognitive ability in males may nottrapolate to females. However, few studies use femalesinvestigate the influence of stress on brain morphology

trogen levels have been found to induce changes in bothCA1 spine density and spine shape in rodents (Gould etal., 1990; Woolley et al., 1997; Li et al., 2004; Gonzalez-Burgos et al., 2005). In addition, ovarian hormones andHRONIC STRESS ENHANCES SPAVARIECTOMIZED FEMALE RATSETRACTION: POSSIBLE INVOLVE

J. MCLAUGHLIN,* S. E. BARAN, R. L. WRIGHT ANDD. CONRAD*

partment of Psychology, Arizona State University, 950 South McAl-r, Box 1104, Tempe, AZ 85287, USA

stractEmerging data report sex differences in how thein responds to chronic stress. Here, we investigated theects of chronic restraint stress (6 h/day/21 days) on hip-campal morphology and function in ovariectomized females. Chronic restraint stress caused CA3 apical dendriticraction in short- and long-shafted neurons, while it re-ced basal dendritic arbors in long-shafted neurons only.ronic restraint did not affect CA1 dendritic arborization,hough it increased the proportion of CA1 spine headsmpared with controls. Both stressed and control animalsrformed well on the Y-maze, a spatial memory task. How-r, chronic stress enhanced Y-maze performance com-red with controls, which may reflect facilitated spatialmory or reduced habituation. Y-maze performance corre-ed with CA1 spine head proportion. This relationship sug-sts that spatial ability in females may be more tightly cou-d with CA1 morphology, which may override the influenceCA3 dendritic retraction. Thus, this research provides ad-ional evidence that CA3 morphology does not always par-l spatial memory. 2005 IBRO. Published by Elsevier Ltd.rights reserved.

y words: hippocampus, chronic stress, females, spatialognition memory, dendritic spines.

xual dimorphisms in the stress response have beenntified for some time, and a growing literature showst stress impacts hippocampal cognitive ability differentlythe two sexes. In humans, stressed males, but notales, exhibit elevated glucocorticoid levels (Kirsch-

um et al., 1992), which negatively correlate with hip-campal-dependent declarative memory (Wolf et al.,01). Similarly in animal models, acute restraint stresspairs hippocampal-dependent spatial memory in male

uroscience xx (2005) xxxstr21spesprothe

rresponding authors. Tel: 1-480-965-2573; fax: 1-480-965-4 (K. McLaughlin), Tel: 1-480-965-7761; fax: 1-480-965-8544D. Conrad).ail addresses: katie.mclaughlin@asu.edu (K. McLaughlin),radc@asu.edu (C. D. Conrad).reviations: ANOVA, analysis of variance; CORT, corticosterone;long-shafted neurons; LTP, long-term potentiation; OVX, ovariec-y/ovariectomized; SS, short-shafted neurons.

6-4522/05$30.000.00 2005 IBRO. Published by Elsevier Ltd. All rights reserved:10.1016/j.neuroscience.2005.06.083

1L MEMORY INPITE CA3 DENDRITICT OF CA1 NEURONS

d cognitive ability, which is alarming given statistics onntal health in humans. Women with a history of long-m depression have a 1015% reduction in hippocampallume compared with controls (Sheline, 1996; Sheline et, 1999) and are almost twice as likely as men to begnosed with depression (Heller, 1993; Kessler et al.,93; Weissman et al., 1993). In animal models, depres-e-like symptoms are often preceded by chronic stress oressful life events, and females cope differently withessful situations than males (Westenbroek et al., 2003).hough recent research has begun to focus on suchnder differences, care and treatment of women hasen derived predominantly from research on males.erefore, more research on females is needed to inves-ate stress effects on the brain and body, and to betterdress womens health.In the brain, stress produces sexually dimorphic re-

onses in both hippocampal morphology and function. Inle rats, restraint for 6 h/day/21 days retracts hippocam-l dendritic arbors in the CA3 region (Watanabe et al.,92c; Magarinos and McEwen, 1995a; Magarinos et al.,98; Conrad et al., 1999), which corresponds to impairedpocampal-dependent spatial memory (Luine et al.,94a,b; Conrad et al., 1996). Pharmacological blockadeCA3 dendritic retraction also correlates with the preven-n of spatial memory deficits in separate studies (Wa-abe et al., 1992a,b; Luine et al., 1994a,b; Conrad et al.,96). In contrast, chronically-stressed, gonadally-intactales have less robust dendritic retraction in basal CA3

ndrites compared with the apical CA3 dendritic retrac-n seen in males (Galea et al., 1997). In addition, spatialmory appears unaffected or improved by chronic stressfemales (Bowman et al., 2001, 2003; Conrad et al.,03; Kitraki et al., 2004). Together, these data suggestt females can have intact or even facilitated spatialmory despite the possible presence of hippocampalndritic retraction.ess appear to have interactive effects on behavior. Afterdays of stress, female rats in proestrus show impairedatial memory compared with rats in other stages of thetrous cycle; however, after 28 days of stress females inestrus show improved spatial memory compared withir control counterparts (Bowman et al., 2001). Through-.

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K. J. McLaughlin et al. / Neuroscience xx (2005) xxx2

ARTICLE IN PRESSt the estrous cycle, the amount of corticosteroneORT) released in response to stress differs, with greateraks in proestrous rats (Viau and Meaney, 1991; Conradal., 2004a). Fluctuations in estradiol and prolactin cano stimulate CORT secretion (Lo and Wang, 2003).erefore, these changes may modulate stress effects onbrain and subsequent behaviors. The interactions

ong stress, ovarian hormones, and hippocampal mor-ology and function are complex; thus, we designed anperiment using ovariectomized (OVX) female rats andute estrogen replacement. This study is the first study toamine the influence of chronic restraint stress and acutetrogen treatment in OVX female rats on both hippocam-l morphology (CA3 and CA1 regions) and hippocampal-pendent spatial recognition memory.

EXPERIMENTAL PROCEDURES

bjects

e Arizona State University Institutional Animal Care and Usemmittee approved all subjects and procedures used for thisearch. All efforts were made to minimize the number of animalsgroup and any potential suffering of these subjects. Twoorts initially totaling 80 female SpragueDawley rats (CDin), weighing approximately 230 g, were purchased fromarles River Laboratories (Hartford, CT, USA). All rats weredomly assigned to either a control (n40) or chronically-ssed group (n40). In addition, rats were administered either estradiol or sesame oil, giving rise to four experimentalditions: control/vehicle (CV), control/estradiol (CE), stressed/icle, (SV) stressed/estradiol (SE). Control and stressed ratsre housed in separate, but identical chambers. Housing cham-s were on a 12-h light/dark schedule (lights on at 7:00 AM) withmperature of 2122 C. Rats were housed two per cage withess to food and water ad libitum.

X

e week after arrival, rats received bilateral ovariectomies. Ratsre anesthetized using a ketamine cocktail (10 ml ketamine, 5 mlazine, 2 ml acepromazine, 3 ml 0.9% NaCl, 1 ml/kg, i.m.). Theomen was rubbed with Betadine surgical scrub (povidone-ine 7.5%) and a longitudinal, 1 cm incision was made to re-ve the ovaries, which were tied off and then cut from theduct. The muscle was sutured with coated Vicryl suture threadhicon, Inc., Somerville, NJ, USA), and the skin was securedh wound clips, which were removed one week post-surgery.

- Estradiol administration

lf of the control and stressed rats received injections (0.1 mL,.) of either 5 g 17- estradiol (Steraloids, Inc., Newport, RI,A) or sesame oil. Injections were given to each rat at 48 h andn again at 24 h prior to behavioral training on the Y-maze, aedule designed to parallel estrogen levels similar to those inestrus (Chesler and Juraska, 2000; Korol and Kolo, 2002). Theeek delay between OVX and estrogen treatment is within the8 week window that acute estrogen replacement has beenviously shown to effectively influence behavior (Becker et al.,7; Frye, 1995; Korol and Kolo, 2002; Markham et al., 2002).ctions coincided with the last two days of restraint, and were

inistered to the animals between 8 and 9 AM before restraint

rted.putUSstraint and handling

ginning 10 days after surgery, half of the rats underwent chronictraint stress for 6 h/day (from approximately 9 AM to 3 PM) fordays. Rats were restrained in their home cages in wire meshtrainers (18 cm circumference24 cm long) and were trans-red to larger restrainers (23 cm circumference28 cm long)en they outgrew the smaller restraint. Control rats remainedisturbed during the time of restraint stress and were housed ineparate but identical chamber.To reduce potential exploration anxiety during the day of

ting, rats were handled for 2 weeks, beginning on day 7 oftraint. All rats were held for one minute, allowed to explore in ae box for approximately twothree minutes, and then wered again for one minute before being returned to their cage.ndling occurred in two different rooms, both separate from theaze room which was used for behavior assessment. To re-e any stress associated with the experimenter, handling wasperformed by researchers who assisted with restraint or withinhours after the restraint session.

aze testing

e Y-maze was used for behavioral testing of spatial recognitionmory. The apparatus consisted of three identical arms (50 l1632 h cm) made of black Plexiglas. The three arms were ran-ly designated as the start, novel, and other arm and werenterbalanced between rats. Animal bedding covered the floorthe maze and was mixed between trials to prevent rats fromng odor cues. Visual cues, in a variety of colors and shapes,re located outside of the maze.The first behavioral trial took place one day after restraintss termination and two hours after the change in light cycle.ts were placed in the start arm and allowed to explore the startother arms for 15 min, while a black Plexiglas divider blockeddesignated novel arm. Four hours later, rats underwent testingwhich they were allowed to explore all arms for 5 min. Theerimenter was not visible to the rats during testing; behaviors videotaped and then quantified at a later date. During quan-ation, the researcher was blind to the arm assignments and theerimental conditions. Entries were defined as entrance of fronts into an arm.

lgi procedure

ts were injected with a lethal dose of Nembutal (1 ml, i.p.), andapitated, then unperfused brains were rapidly removed. FDpid Golgistain kits (FD NeuroTechnologies, Baltimore, MD,A) were used for Golgi staining, and staining was successful forcohort of rats. Brains were prepared as directed by the kit and

n quickly frozen in 2-methylbutane and cut in 100 m sectionsicrotome HM 500 OM Cryostat, 30/32 C). Once placed on ae, brains were firmly pressed by hand using Bibulous blottinger (Fisher Scientific International Inc., USA). Brains were pro-sed, left in the dark to dry for 12 weeks, stained according toGolgistain Kit, and then coverslipped with Permount Mount-Media (Fisher Scientific International Inc.).

tology

r proper Golgi analysis, cells were chosen based on the follow-criteria: 1) the cell body and dendrites were fully impregnated,he cell was relatively isolated from surrounding neurons, and 3)cell was located in either the CA3c or CA1 region of the

pocampus. A camera lucida drawing tube attached to an Olym-BX51 microscope was used to trace all neurons (320).

ndritic length was quantified using a Scion Image Microcom-

er Imaging Device Program (Scion Corporation, Frederick, MD,A) attached to an Olympus BX 50 microscope. Branch points of

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K. J. McLaughlin et al. / Neuroscience xx (2005) xxx 3

ARTICLE IN PRESSdendritic tree were also counted. The neurons used for anal-s were randomly selected with the only restriction being thatple sizes were equal between the groups.

CA3. Six rats from each experimental group (total of 24s) were chosen in which sufficient numbers of Golgi-impreg-ed cells could be identified. CA3 neurons were further labeledshort-shaft (SS) or long-shaft (LS) depending on their relativeation in the stratum pyramidale and proximal apical shaft lengthtch et al., 1989). Dendritic length and branch points were mea-ed separately for the apical and basal sections of the neuron.r each rat, apical analyses included two SS and two LS neuronsraged to obtain one value for SS and LS cells, respectively.sal analyses were performed similarly, with three SS and threecells separately averaged for each rat.

CA1. A total of five rats from each group (total of 20 rats)re chosen in which sufficient Golgi staining was present. Forh rat, six neurons were analyzed. CA1 cells also express tworonal types; however, cell complexity does not differ like CA3ls (Bannister and Larkman, 1995). Therefore, both cell typesnd in the CA1 region were analyzed together and averaged toain one measure of branch points and one measure of branchgth per rat.CA1 spine densities from pyramidal cells that had consistent

lgi impregnation were also analyzed because spines fluctuateoss the estrous cycle (Woolley et al., 1990; Shors et al., 2001)estrogen is one of the most robust modulators of spine density

he CA1 region (Shors et al., 2001). Three cells each from fives in each group (total of 20 rats) were chosen randomly forne analyses. Following previously established protocols (Gouldal., 1990; Woolley et al., 1997), spines from the most lateralcal tertiary dendrite and the most lateral basal secondary den-e were quantified. A camera lucida drawing tube was used toce spines that could clearly be seen in one focal field (1250).ines were then counted, and the density was calculated as theber of spines/10 m of dendrite. There were visible differ-es between CA1 spine shapes. In general, young hippocampalnes are headless, and exhibit heads as they mature, usuallyr 24 weeks in vitro (Papa et al., 1995). Therefore, spines withds were differentiated from those without heads, and we sep-tely compared the proportion of each with total dendriticnes.

tistical analyses

ree rats were removed due to surgical complications or equip-nt errors, and the final analyses for the Y-maze included 77 ratsntrol n39, stress n38).

Golgi. An analysis of variance (ANOVA) with treatmentntrol, stress) and hormone (vehicle, estradiol) as the indepen-t variables was used to analyze the effects on CA3 and CA1rphology. Separate comparisons were made for apical andal branch points and length. For CA3 analyses, a within-ject design for cell type (SS, LS) was employed. For CA1lyses, an ANOVA with spine density as the dependent variables computed. Since CA1 dendritic spines were represented asios of dendritic spines with heads to total dendritic spines, thene density data were transformed according to the equation forportions, 2arcsin (square root (Y)) (Cohen et al., 2003).

Y-maze. To assess spatial memory within a given condi-, a Wilcoxon non-parametric matched pairs test was used topare entries into the novel and other arms. The start arm wasincluded in this analyses because rats were initially placedre by the experimenter, potentially creating a bias againstentering that arm. Differences in spatial memory across groups

re determined by ANOVA using treatment (control, stress) andmone (vehicle, estradiol) as the independent variables. The

F(1Pendent variable was a difference score, generated by subtract-the percentage of entries into the other arm from the percent-of entries into the novel arm. Positive scores reflect a prefer-e for the novel arm, while negative scores indicate a prefer-e for the other arm. A zero score indicates chanceformance with no arm preference. Total entries (sum of entriesall three arms) were also used to determine whether motor ortivational factors differed among the groups (Conrad et al.,6).To assess the relationship between morphology and behav-Spearman correlations (one-tail, P0.05) were performed topare CA3 dendritic properties or CA1 spines with total entries

difference scores.

Body weights. An ANOVA with treatment (control, stress)hormone (vehicle, estradiol) was the between-subjects vari-

e and day (1, 7, 14, and 21) as the within-subject variable wasd to determine the effects of stress or estrogen on bodyight.

Software. The statistical software program STATISTICAatsoft Inc., Tulsa, OK, USA) was used for all ANOVAs andarson correlations. Significant effects from ANOVA (P0.05)re further analyzed using Newman-Keuls post hoc tests, unlesserwise noted. Spearman correlations were conducted usingSS (SPSS Inc., Chicago, IL, USA, version 12.0).

RESULTS

trogen treatment failed to produce statistically significantects on morphology (P0.10), behavior (P0.10), oright (P0.1). Therefore, the data were collapsed acrosshicle- and estrogen-treated groups, and only analysesated to stress effects are reported. The final number ofs per group was the following for morphology: CA3ntrol n12, stress n12), CA1 (control n10, stress10), CA1 spines (control n10, stress n10).

ects of chronic stress on hippocampalrphology

CA3 apical dendritic arborization. Chronically-stresseds displayed apical CA3 dendritic retraction (Fig. 1). A2 mixed-factor ANOVA (treatmentcell type) revealedsignificant main effect of treatment for apical branchints, F(1,22)11.40, P0.01, and total dendritic branchgth, F(1,22)5.22, P0.05. Restrained rats expresseder branch points and less total branch length than

ntrols (Fig. 2A, B). As expected, a significant effect ofll type was also demonstrated, with SS cells havingre branch points, F(1,22)9.35, P0.01, and greatererall branch length, F(1,22)4.47, P0.05, comparedh LS cells, regardless of treatment. All other effectsre not significant (P0.10). Pearson correlations faileddemonstrate any significant relationships between CA3ical branch points and length and difference scores,0.10

CA3 basal dendritic arborization. Chronic stress re-ced branch points and length of LS, but not SS, basalors (Fig. 2C, D). A 22 mixed-factor ANOVAatmentcell type) indicated a significant interaction be-en treatment and cell type for branch points,

,22)9.59, P0.01, and branch length, F(1,22)5.96,0.05. Further analyses using separate one-way ANO-

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K. J. McLaughlin et al. / Neuroscience xx (2005) xxx4

ARTICLE IN PRESSs for the control and stress conditions demonstratedt indeed, chronic restraint specifically decreased LSsal dendritic properties without affecting SS basal den-

. 1. Golgi-Stained CA3 Neurons. Camera lucida drawings (320)resent experimental conditions of the two CA3 neuronal typesdied. (A) Control SS, (B) Stress SS, (C) Control LS, and (D) StressNote the reduction in apical dendritic complexity in the stressed (B,vs. control neurons (A, C). Basal dendritic retraction was evident inLS stressed group only (D) compared with LS control neurons (C).

. 2. CA3 apical and basal dendritic morphology. Chronic restraint deccells had more apical branch points and overall apical branch lengthtraint reduced both LS basal branch points (C) and length (D) co

ansS.E.M. * P0.05 stress vs control. P0.05 SS vs. LS. For basal data12 rats/group).tes. All other effects were not significant (P0.10). Ad-ionally, Pearson correlations failed to demonstrate anynificant relationships between CA3 basal dendritic prop-ies and differences scores, P0.10.

CA1 apical and basal dendritic arborization. CA1 api-l and basal dendritic arborization were unaffected by theatment condition. There were no significant main effectsinteractions for CA1 branch points or branch lengths0.10; Table 1).

CA1 dendritic spines. A one-way ANOVA for treat-nt showed no significant effects for apical (groups means:trol9.77, stress10.80.7) or basal CA1 spine den-es (control9.80.7, stress9.80.8, Ps0.10). How-er, chronic stress significantly influenced basal spineape (Fig. 3). Chronically restrained rats exhibited a sig-cantly greater proportion of basal spines with headsmpared with controls, F(1,18)8.56, P0.01. Chronicess did not alter spine shape on apical dendrites800.02, 0.840.04, for proportion of spines with

oth the apical branch points (A) and branch length (B). In addition,ed with LS cells for both stressed and control conditions. Chronicwith SS branch points and length. Data points represent group

le 1. CA1 apical and basal dendritic morphology (meansS.E.M.)

Apical Basal

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K. J. McLaughlin et al. / Neuroscience xx (2005) xxx 5

ARTICLE IN PRESSads in control and stress groups, respectively). No otherects were significant, P0.10.

ects of chronic stress on the Y-maze

one-way ANOVA for treatment showed no statisticalference for total entries between groups for the full fivenutes, P0.05. However, a recent study demonstratedinteraction between sex and minute for entries, with theatest locomotor activity for female rats occurring withinfirst minute (Conrad et al., 2003). Due to this precedent

d a possible indication of female habituation to the Y-maze,wo-way mixed-factor ANOVA (treatmentminutes) wasducted to analyze behavior in the first minute comparedh the subsequent four minutes. All groups made signif-ntly more entries during the first minute compared withsubsequent minutes, F(1,75)104.80, P0.001. In ad-

ion, restrained rats made significantly fewer entries50.2) than their control counterparts (4.20.2) duringfirst minute, F(1,75)5.14, P0.05.All groups entered the novel arm significantly moren the other arm during minute 1, according to a Wil-xon matched pairs test (P.01; Fig. 4A). Moreover, a

. 3. Basal CA1 dendritic spine proportions (heads to total). (A)mera lucida drawings (1250) represent CA1 basal dendriticnes from control and stress groups. The closed arrow points to anmple of a headless spine whereas the open arrow points to a spined. (B) Stressed rats had significantly more basal dendritic spinesheads compared with controls. Data points represent group

ansS.E.M. * P0.05 (n10 rats/group).e-way ANOVA for treatment showed a significant effect,,75)4.32, P0.05, with chronically restrained rats ex-

trospiting higher positive difference scores compared withntrols (Fig. 4B).Spearman correlations performed between CA3 den-

tic retraction and behavior showed no statistically signif-nt results for morphological correlations with total en-s or difference scores, P0.10. However, a correlationtween CA1 basal spine head proportion (spines withads/total spines) and difference score on the Y-maze

demonstrate a significant, positive relationship,8)0.54, P0.05. As difference scores became moresitive, the proportion of spine heads increased. Thejority of rats with the greatest difference scores andine heads represented rats from the restrained groupg. 5). There were no other significant effects, P0.10.

dy weights

predicted, restrained rats gained weight more slowlyn controls over 21 days of chronic restraint. A 24xed-factor ANOVA (treatmentday) revealed statisti-lly significant main effects of treatment, F(1,77)72.28,0.001 and day, F(3,231)212.6, P0.001 and a signif-nt interaction between treatment and day, F(3,231).44, P0.001. Stressed females gained less weight overdays of restraint (268.92.4 g, 291.73.5 g, for days 1d 21, respectively) compared with non-restrained con-

. 4. Influence of chronic stress on Y-maze performance. (A) Bothups entered the novel arm more than the other arm,0.05 Nvel vs. Other. (B) Stressed rats had significantly greateritive difference scores compared with control rats, * P0.05, stresscontrol. Data points represent group meansS.E.M. (control

39, stress n38).ls (272.82.8 g, 329.53.1 g, for days 1 and 21, re-ectively).

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K. J. McLaughlin et al. / Neuroscience xx (2005) xxx6

ARTICLE IN PRESSDISCUSSION

is experiment investigated the effects of chronic stresshippocampal morphology and spatial memory in OVXale rats. The results indicate that CA1 spine shape

lowing chronic stress may parallel spatial memory inale rats despite robust CA3 dendritic retraction. More-

er, spatial memory can be intact or perhaps even en-nced in situations in which hippocampal CA3 dendriticraction is present. Consequently, this study did not sup-rt the global interpretation that CA3 morphology predictsatial memory, as demonstrated in male rats. Althoughmerous studies have suggested that ovarian hormonesntribute to functional spatial memory in stressed femaleswman et al., 2002; Bisagno et al., 2003), we havemonstrated that ovarian hormones are not necessary foract spatial memory. Overall, these findings suggest thatCA1 region may play an important role in spatial mem-ability and that it may override the contribution of the3 region following chronic stress in females.

ects of chronic stress on hippocampalrphology

CA3. A previous study using intact females demon-ated CA3 dendritic retraction in the basal, but not apicalion (Galea et al., 1997). In contrast, OVX females in therrent study showed apical dendritic retraction in the CA3ion, which was consistent across both measures ofnch points and total branch length. This apical dendriticraction was similar to that seen in previous researching males (Watanabe et al., 1992c; Magarinos and McE-n, 1995a; Galea et al., 1997; Conrad et al., 1999; Sousaal., 2000; Vyas et al., 2002). Perhaps OVX enabled CA3ical dendritic retraction to occur by the removal of anytential neuroprotective effects of estrogen (Simpkins et, 1997; Lee and McEwen, 2001; Bisagno et al., 2003).e current study also supported previous data thatronic stress reduced CA3 basal arbors in females (Ga-

. 5. Correlation between Y-maze difference scores and proportionA1 basal spine heads. There was a significant, positive relation-

p for greater difference scores on the Y-maze (i.e. preference fornovel arm) to be associated with higher proportions of CA1 den-ic spines with heads, rs(8).54, P0.05 (n10 rats/group).et al., 1997). In addition, we present novel findings thatess-induced CA3 basal dendritic retraction is carried

minudominantly by LS and not SS neurons. This uniqueture of stress targeting one cell type in females has alsoen observed in males, but with SS apical dendritesing more susceptible to stress than LS apical arborsmbert et al., 1998). These current findings indicate thatving a bias for one cell type could potentially skewults, which may account for the discrepancy betweendies. An alternate explanation for the discrepanciesong studies (Galea et al., 1997) could be differences inlgi methodology, but this interpretation is mitigated forveral reasons. First, potential artifacts should be similartween experimental treatments. Second, our consistentasures of dendritic retraction derived from branchints and total branch length support our interpretationd confirm that SS cells are more complex than LS cellstch et al., 1989). Finally, observing CA3 dendritic retrac-n using different Golgi procedures lends support thatronic stress alters hippocampal morphology.

CA1. Chronic stress did not affect spine density, butincreased the proportion of spine heads in the CA1ion. Spines change shape as they mature, most notice-ly by developing a head at the end of the spine (Kasai et, 2003). Large, more mature spines express high num-rs of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepro-onic acid receptors, greater glutamate sensitivity, andassociated with long-term potentiation (LTP), which isught to reflect greater synaptic activity and memoryction (Matsuzaki et al., 2001; Kasai et al., 2003). Indition, drugs that inhibit actin filaments within spineads interfere with LTP, which is thought to be importantmemory coding and consolidation within the hippocam-s (Mesches et al., 1999; Ackermann and Matus, 2003).ile ovarian hormones and androgens induce changes inth spine density and shape in rodents (Gould et al.,90; Woolley et al., 1997; Leranth et al., 2004; Li et al.,04; Gonzalez-Burgos et al., 2005), these data show thatronic stress modulates CA1 spine shape as well.

ects of chronic stress on the Y-maze

ronically-stressed rats showed greater novel arm pref-nce compared with controls on the spatial recognitionmory component of the Y-maze. Stress may have en-nced performance by interfering with habituation to thevel arm. This interpretation is supported by impairedbituation to novel environments in male rats followingychosocial stress (Park et al., 2001). Another interpre-ion is that chronic stress facilitated females spatialmory, similar to the facilitative effects found in the radialmaze and object placement tasks (Bowman et al.,

01, 2002, 2003; Beck and Luine, 2002). This diversity ofks that use novelty or appetitive training suggests thatrformance in females is facilitated by chronic stressough neural mechanisms underlying spatial memory.Non-mnemonic mechanisms, such as changes in mo-ability or motivation, may have also contributed to theilitated performance of stressed females. During the five

nutes of testing, control and stressed rats made a similarmber of entries in the Y-maze, suggesting that both

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K. J. McLaughlin et al. / Neuroscience xx (2005) xxx 7

ARTICLE IN PRESSups had similar motor behavior and motivational ability.thin minute 1, however, stressed females made fewertries than controls. These data indicate that fewer en-s are not necessarily synonymous with reduced motorility or motivation because rats showing the best novelpreference made the fewest entries.Another interpretation is that stress influenced the pref-nce for novelty and altered performance in the Y-mazeaffecting entries into the novel arm. However, male ratst are impaired in the traditional version of the Y-mazeccessfully seek out the novel arm when salient, intrinsices are provided or when the intertrial-interval is reduced1 min (Wright and Conrad, 2005; Kleen et al., 2004).ether chronic stress enhances novelty-seeking behav-in females has yet to be determined.Differences in the perception of restraint betweenles and females may explain the lack of impairment inale spatial memory ability. Some reports have shownt males have less weight gain following chronic stressn females (Watanabe et al., 1992c; Magarinos andEwen, 1995b; Conrad et al., 2003, 2004b), which sug-sts that males may perceive restraint as more stressfuln females. However, the females in the current studyplayed robust CA3 hippocampal dendritic retraction inth the apical and basal regions of the hippocampus,ich is more extensive than the amount of stress-induced3 dendritic retraction previously reported for males us-the same restraint protocol (Watanabe et al., 1992c;garinos and McEwen, 1995a; Conrad et al., 1999).erefore, the females in our study appear to have per-ived restraint as stressful as males.Neurochemical and hormonal changes following OVX

d/or chronic stress may have contributed to spatialmory performance. Young and aged rats who experi-ce long-term OVX (1.56 months) display significantlytter performance in the water radial-arm maze than theiram counterparts, most likely from the hormonal shiftcurring after OVX (Bimonte-Nelson et al., 2003). Chronictraint stress affects the dopaminergic system in thefrontal cortex, and these stress-dependent changes aret demonstrated in gonadally-intact stressed femaleswman et al., 2002). Three weeks following OVX, fe-les have a faster recovery of adrenocorticotropin hor-ne (ACTH) and CORT response to shock stress com-red with females given chronic estrogen replacementrgess and Handa, 1992). In general, females haveher basal levels of CORT and release more CORT thanles in response to stress (Atkinson and Waddell, 1997;lea et al., 1997; Rivier, 1999; Kitraki et al., 2004). At theme time, female rats habituate more quickly to restraintess compared with males (Bowman et al., 2001; Luine,02), but these sex differences in CORT production aret always observed (Figueiredo et al., 2002; Conrad et, 2004a). In addition, chronic stress sex-dependentlyers the two CORT receptors within the CA1 region of thepocampus (Kitraki et al., 2004). Any one or combinationthese changes may influence the success of chronically

essed females in spatial memory tasks. Moreover, these

tactioctuations may affect other areas in the brain, in additionhippocampal CA3 and CA1 cells.

e lack of an estrogen influence on hippocampalrphology and behavior

e similarity of CA1 spine densities across experimentalups may seem surprising, as it has been well docu-nted that acute estrogen treatment increases apical1 spine density (Woolley and McEwen, 1993; Woolleyal., 1997). Within 48 h of administering 17-estradiol,1 spine density increases and these maximal levels areintained for an additional 48 h after estrogen is metab-ed, then density slowly declines over the next few daysoolley and McEwen, 1993; Woolley, 1998). While the-estradiol dose used in this study (0.1 mL of 5 g) isilar to that which has been used in other behavioralradigms (Chesler and Juraska, 2000; Korol and Kolo,02), this dose is too low to fully reproduce increases in1 apical spine density which have been demonstratedmorphological studies (0.1 mL of 10 g; Gould et al.,90; Woolley and McEwen, 1993; Woolley, 1998). There-e, estrogen treatments used in future research will needinclude higher doses in order to determine a more clearationship between changes in the brain and subsequenthavior. In addition to dose, differences between pastearch and the present study may involve a difference inhavioral training and handling procedures. For example,nificant differences in CA1 spine densities between es-gen and vehicle treated rats disappear following han-g for 5 min/day/14 days (Garza-Meilandt et al., 2002).us handling, combined with estrogen replacement, mayve retarded OVX-induced changes in spine density inr rats.Estrogens inability to alter Y-maze performance

ould also be further explored. Acute estrogen replace-nt can be given 3 weeks to 4 months after OVX toserve changes in memory (Korol and Kolo, 2002; Sand-om and Williams, 2004). Administering 17-estradiol toX females either 72 and 48 h, or 48 and 24 h beforehavioral assessment, which is consistent with our timeme, can either improve or impair spatial memory, de-nding on the task, compared with controls (Frye, 1995;ndstrom and Williams, 2001; Korol and Kolo, 2002).rhaps our handling procedure may have negated estro-ns effects on the Y-maze because of the enriched set-g. Environmental enrichment can increase spatial mem-ability on several tasks (Fernandez-Teruel et al., 1997;

esack and Frick, 2004). However, a recent finding dem-strated that while mice treated with estrogen had im-ved spatial memory, estrogen combined with previousposure to an enriched environment did not alter objectognition and actually impaired work memory acquisitionresack and Frick, 2004). Although this study used miced an enriched environment protocol lasting severalnths, the results provide an interesting possibility. Sinceof the rats in this study were exposed to the samendling and environmental cues and all animals had in-

t spatial memory, we cannot separate possible interac-ns between estrogen and the environment.

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K. J. McLaughlin et al. / Neuroscience xx (2005) xxx8

ARTICLE IN PRESSnificance

r research contributes to growing evidence demonstrat-that CA3 hippocampal morphology does not alwaysdict spatial memory function. Although CA3 dendriticraction is associated with memory deficits in males,ronically-stressed male rats exhibit increased hippocam-l-dependent contextual fear conditioning compared withntrols, despite CA3 dendritic retraction (Conrad et al.,99, 2001). In addition, inhibiting CORT synthesis withtyrapone on the day of training prevents stress-inducedatial memory deficits when dendritic retraction should besent (Wright et al., 2004). Finally, three weeks of CORTatment causes CA3 dendritic retraction, but spatialmory remains intact on the Y-Maze (Coburn-Litvak et, 2003). Therefore, CA3 dendritic retraction does notays parallel spatial memory deficits in males, and ourdings with females contribute to this growing literature.r study indicates that stress-induced changes in CA1ine shape are associated with enhanced spatial memoryVX female rats. These data are consistent with studies

owing that changes in CA1 spines, following estrogenatment, parallel spatial memory retention (Sandstromd Williams, 2004). Together these findings suggest thatpresence of mature spine heads on CA1 hippocampal

ndrites may play a more critical role than CA3 morphol-y in predicting spatial memory in females.

knowledgmentsThis work was funded by MH64727 (Conrad).e contributions of following individuals are gratefully acknowl-ed: Rudy Bellani, Meghan Ellis, Juan Gomez, Gillian Hamilton,es Harman, Jonathan Kleen, Jamie Jackson, Scott Seganti,

rgey Tsekhanov, and Lindsay Wieczorek.

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(Accepted 16 June 2005)

CHRONIC STRESS ENHANCES SPATIAL MEMORY IN OVARIECTOMIZED FEMALE RATS DESPITE CA3 DENDRITIC RETRACTION: POSSIBLE INVOLVEMENT OF CA1 NEURONSEXPERIMENTAL PROCEDURESSubjectsOVX17- Estradiol administrationRestraint and handlingY-maze testingGolgi procedureHistologyCA3.CA1.

Statistical analysesGolgiY-mazeBody weightsSoftware

RESULTSEffects of chronic stress on hippocampal morphologyCA3 apical dendritic arborizationCA3 basal dendritic arborizationCA1 apical and basal dendritic arborizationCA1 dendritic spines

Effects of chronic stress on the Y-mazeBody weights

DISCUSSIONEffects of chronic stress on hippocampal morphologyCA3.CA1.

Effects of chronic stress on the Y-mazeThe lack of an estrogen influence on hippocampal morphology and behaviorSignificance

AcknowledgmentsREFERENCES